1. Field of the Invention
The present invention relates to a process for the lyophilization of acid producing bacteria and to the lyophilized products. In particular the present invention relates to the use of: (1) a substantial amount of an inorganic or organic buffering agent for the lyophilization; and (2) an oxygen free gaseous atmosphere or vacuum for the bacteria in order to make them storage stable.
2. Prior Art
Lyophilization or vacuum freeze-drying of heat sensitive compositions is well known to the prior art. The process is generally described in Kirk-Othmer Vol. 7, pages 368 and 369, (1965) and in the literature. The freezing-drying equipment used in the process includes: (1) freezing equipment; (2) a vacuum drying chamber; (3) a vacuum source; (4) an optional heat source and (5) a vapor-removal system. The ice in the frozen liquid sublimes and leaves a porous powdered composition.
Alkali metal salts of glycerophosphate have been suggested for use in enhancing the growth of bacteria by Douglas in Laboratory Practice 20: 414-417 (1971) and 23:3-5 (1974). In U.S. Pat. No. 3,975,575, one of the inventors herein described frozen concentrates of lactic acid forming bacteria incorporating an alkali metal salt of glycerophosphoric acid which have enhanced milk fermenting activity when thawed and used. In our copending application Ser. No. 649,853 filed Jan. 16, 1975 now abandoned, we described the enhancement of the milk fermenting activity of previously freeze-dried lactic acid producing bacterial cells by the addition of an alkali metal salt of glycerophosphoric acid to the freeze-dried bacteria. The glycerophosphates are used in amounts up to about five (5%) percent by weight of the bacterial concentrate. There has been no suggestion by the prior art that the salts of glycerophosphoric acid might be used for the lyophilization.
It is well known that the freeze-drying of bacteria kills most of them, usually on the order of ninety-nine percent. Those that survive die rapidly upon storage. Attempts have also been made by the prior art to increase the number of surviving bacteria upon lyophilization and storage with limited success. For instance U.S. Pat. No. 3,897,307 and an article by Speckman et al in Journal of Dairy Science Vol. 57, No. 2 (1973) describe such processes. Various chemical additives are used for the lyophilization and sometimes a vacuum in an attempt to increase the viability upon lyophilization and storage. Usually a storage time of at least about ninety (90) days with a significant survival of the bacteria on a percentage basis is needed for commercial purposes.
The rare gases have been generally used as a covering atmosphere to protect materials from the air or from moisture. These gases include helium, neon, krypton, and argon. Argon is derived from air and can be made essentially free of oxygen by passage of impure argon over heated copper; by selective adsorption on synthetic zeolites; or by the addition of hydrogen followed by catalytic combustion to remove oxygen and reliquefication of the argon to remove excess hydrogen. The resulting argon is 99.99 plus percent by weight pure and thus is essentially free of oxygen. It can be highly purified easily and relatively inexpensively because it is very inert in comparison to oxygen.